G protein-coupled receptors (GPCRs) transmit signals from a diverse array of external stimuli. The cell surface density of GPCRs critically dictates both quantity and quality of GPCR signaling. The cell surface expression of GPCRs is spatio-temporally regulated by dynamic interactions involving discrete sorting motifs on the cargo and intracellular protein machineries. Naturally, genetic defects in any of these interactions result in the intracellular retention of GPCRs that cause human diseases. Increasing evidence suggests the essential role of an RXR (di-basic, di-Arg) motif in the quality control of membrane proteins by regulating their endoplasmic reticulum (ER)-to-Golgi forward transport. However, molecular basis for the role of RXR motif in regulating the cell surface density of GPCRs is very poorly understood in spite that a large number of GPCRs carry potential RXR motif. Our overall aim is to understand the molecular basis for the maturation and signal transduction of GPCRs. Under this broad objective, in this grant proposal we will focus on elucidating the molecular mechanisms by which a RXR motif regulates the cell surface density of GPCRs in multiple trafficking pathways by studying an HIV co-receptor GPR15 as a model protein. We have recently demonstrated that an RXR motif plays a key role in the ER retrieval/retention of GPR15 in the absence of a phosphorylation-dependent 14-3-3 protein to the receptor C-terminus. This activity is mediated by the interaction with a retrograde coatomer complex COPI. Our study has now revealed a novel role of RXR motif in promoting the signal-induced endocytosis of GPR15 from the cell surface. We have also found that the RXR motif activity is regulated by a serum- and glucocorticoid-regulated kinase (SGK1) that phosphoylates GPR15 C-terminus and recruit 14-3-3 proteins. Furthermore, we have designed a unique yeast-based functional screening system to develop a peptide-based approach that attempts to modulate the RXR motif activity. Based on these observations, we hypothesize that an RXR motif plays a key role in regulating the cell surface density of GPCR in both constitutive and signal-induced trafficking through dynamic interactions with transport machineries, and that these interactions can be modulated by specific signaling pathways and small molecules. To test these hypotheses we will investigate following specific aims: (1) To determine a novel role of RXR motif in the endocytosis of GPR15, (2) To define the kinase signaling pathways that regulate RXR activity and trafficking of GPR15, and (3) To develop a peptide-based approach to specifically modulate RXR motif activity and cell surface expression of GPCRs. Overall, these studies will reveal novel molecular mechanisms by which the cell surface density of GPCRs is regulated. The information obtained from these studies will also provide important technological basis for treating the diseases involving abnormal trafficking and functioning of GPCRs.